The study of gene expression has required the development of genes which are relatively easy to assay and can serve as markers to monitor expression of foreign genetic material which has been introduced into cells. The foreign genomic DNA fragments are inserted into a recombinant plasmid along with a marker gene, and after introduction of the plasmid into the target cell, the quantity of protein coded for by the marker gene is determined. When compared to the quantity of protein produced by a plasmid without the insert, the result is indicative of the activity of the inserted fragment.
Marker genes which have been used for this purpose fall into two main categories: those which code for proteins which are detected by antibodies and those which code for proteins which are detected by their enzyme activity. Genes in the first category require the production of specific antibodies to the protein and development of a suitable detection method for measurement of antibody-protein binding. The second group includes genes which code for enzymes such as betagalactosidase, glucuronidase, thymidine kinase, and chloramphenicol acetyltransferase (CAT).
Enzyme marker genes have the advantage of being easy to assay by simply measuring enzyme activity in the cellular extract, while immunoassay-based systems require the development of specific antibodies and an immunoassay technique which may be complex and tedious. However, many enzyme assays are subject to background interference by endogenous enzyme activity in the cells being measured. The CAT gene from bacteria is not normally found in mammalian cells and therefore offers the benefit of a simple, sensitive enzyme-based assay which is free from background interference.
Use of the CAT gene as a marker for measuring promoter function in transfected recombinants was first described by Gorman, et. al, "Recombinant Genomes which Express Chloranphenicol Acetyltransferase in Mamalian Cells," Mol. Cell Biol. 2:1044 (1982). In this work, CAT enzyme activity in cell extracts was measured by extracting the [.sup.14 C]chloramphenicol mixture into an organic solvent (ethyl acetate) and separating the mono- and di-acetates by thin-layer chromatography on silica gel. After autoradiography of the separated chloramphenicol derivatives, the spots were cut from the plates and counted to give a quantitative estimate of CAT activity. Other investigators [Young, et. al., "Detection of Acetyltransferase Activity in Transfected Cells: A Rapid and Sensitive HPLC-Based Method," DNA 4(6):469 (1985) and Burzio, et. al., "Assay of Chloramphenicol Acyltransferase by High-Performance Liquid Chromatography, Gene Anal. Techn. 5:5 (1988)]have measured CAT activity by HPLC of the organic extract and claim sensitivity equivalent to the original TLC method with the added advantage of a substantial reduction in processing time and the elimination of radioactive materials. A third method for assaying CAT was reported by Neumann, et. al., "A Novel Rapid Assay for Chloramphenicol Acetyltransferase Gene Expression," Biotechniques 5(5):444 (1987). Using [.sup.3 H]acetyl Coenzyme A (which transfers acetyl to chloramphenicol in the enzymic process), the cell incubation was performed in a scintillation vial overlaid with scintillation cocktail. Under these conditions, the only radioactive product that diffuses into the cocktail is acetylated chloramphenicol, allowing kinetic analysis through direct measurement of the radioactivity without requiring a manual separation step. This method is fast and convenient, but requires expensive and hazardous radioactive materials of high specific activity.
The use of fluorescence as a means of detection in enzyme assays and immunoassays is recognized to provide many advantages over methods that employ spectrophotometry or measurement of radioactivity for detection [Gerson, "Fluorescence Immunoassay," J. Clin. Immunoassay 7(1):73 (1984)]. Among these advantages is the elimination of the danger and expense of handling radioactive materials and the higher potential sensitivity afforded by measurement of a fluorescent signal as compared to spectrophotometry. Higher sensitivity provides, in turn, the potential advantages of greater accuracy and faster assay times.